This invention relates generally to contrast agents for ultrasound diagnosis and a process for making the same. In particular, the present invention relates to a microbubble composition comprising a lipid shell, a stabilizer coated on the lipid shell and perfluoropropane gas encapsulated in the lipid shell. The lipid shell comprises 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, monosodium salt, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2 dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, monosodium salt and the stabilizer comprises a high molecular weight polymer. In the microbubble composition of the invention, the lipid component is not conjugated to the stabilizer.
Ultrasound is a valuable diagnostic imaging technique for studying various areas of the body, for example, the vasculature, including tissue microvasculature. Ultrasound provides certain advantages over other diagnostic techniques. For example, diagnostic techniques involving nuclear medicine and X-rays generally results in exposure of the patient to ionizing electron radiation. Such radiation can cause damage to subcellular material, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. Ultrasound does not involve such potentially damaging radiation. In addition, ultrasound is relatively inexpensive relative to other diagnostic techniques, including computed tomography (CT) and magnetic resonance imaging (MRI), which require elaborate and expensive equipment.
Ultrasound involves the exposure of a patient to sound waves. Generally, the sound waves dissipate due to absorption by body tissue, penetrate through the tissue or reflect off of the tissue. The reflection of sound waves off of tissue, generally referred to as backscatter or reflectivity, forms the basis for developing an ultrasound image. In this connection, sound waves reflect differentially from different body tissues. This differential reflection is due to various factors, including the constituents and the density of the particular tissue being observed. Ultrasound involves the detection of the differentially reflected waves, generally with a transducer that can detect sound waves having a frequency of one megahertz (Mhz) to ten Mhz. The detected waves can be integrated into an image which is quantitated and the quantitated waves converted into an image of the tissue being studied.
Ultrasound imaging techniques generally also involve the use of contrast agents. Contrast agents are used to improve the quality and usefulness of images which are obtained via ultrasound. Exemplary contrast agents include, for example, suspensions of solid particles, emulsified liquid droplets, and gas-filled bubbles. See, e.g., Hilmann et al., U.S. Pat. No. 4,466,442; Unger, U.S. Pat. No. 5,088,499; Unger, U.S. Pat. No. 5,547,656; Unger and Wu, U.S. Pat. No. 5,228,446; Unger et al., U.S. Pat. No. 5,715,824; Unger and Wu, U.S. Pat. No. 5,769,080; Unger, U.S. Pat. No. 5,705,187; Unger et al., U.S. Pat. No. 5,773,024; Unger et al., U.S. Pat. No. 5,535,112; and published International Patent Applications WO 92/17212 and WO 92/21382.
The quality of images produced from ultrasound has improved significantly. Nevertheless, further improvement is needed, particularly with respect to images involving vasculature in tissues that are perfused with a vascular blood supply. Accordingly, there is a need for improved ultrasound techniques, including improved contrast agents which are capable of providing medically useful images of the vasculature and vascular-related organs.
The reflection of sound from a liquid-gas interface is extremely efficient. Accordingly, bubbles, including gas-filled bubbles, are useful as contrast agents. Exemplary bubbles include, for example, liposomes, micelles and the like. As discussed more fully hereinafter the effectiveness of bubbles as contrast agents depends upon various factors, including, for example, the size and/or elasticity of the bubble.
With respect to the effect of bubble size, the following discussion is provided. As known to the skilled artisan, the signal which is reflected from a bubble is a function of the radius (r6) of the bubble (Rayleigh Scatterer). Thus, a bubble having a diameter of 4 micrometer (xcexcm) possesses about 64 times the scattering ability of a bubble having a diameter of 2 xcexcm. Thus, generally speaking, the larger the bubble, the greater the reflected signal.
However, bubble size is limited by the diameter of capillaries through which the bubbles must pass. Generally, contrast agents which comprise bubbles having a diameter of greater than 10 xcexcm can be dangerous since microvessels may be occluded. Accordingly, it is desired that greater than about 99% of the bubbles in a contrast agent have a diameter of less than 10 xcexcm. Mean bubble diameter is important also, and should be greater than 1 xcexcm, with greater than 2 xcexcm being preferred. The volume weighted mean diameter of the bubbles should be about 7 to 10 micrometer.
As noted above, the elasticity of bubbles is also important. This is because highly elastic bubbles can deform, as necessary, to xe2x80x9csqueezexe2x80x9d through capillaries. This decreases the likelihood of occlusion. The effectiveness of a contrast agent which comprises bubbles is also dependent on the bubble concentration. Generally, the higher the bubble concentration, the greater the reflectivity of the contrast agent.
Another important characteristic which is related to the effectiveness of bubbles as contrast agents is bubble stability. As used herein, particularly with reference to gas-filled bubbles, xe2x80x9cbubble stabilityxe2x80x9d refers to the ability of bubbles to retain gas entrapped therein after exposure to a pressure greater than atmospheric pressure. To be effective as contrast agents, bubbles generally need to retain greater than 50% of entrapped gas after exposure to pressure of 300 millimeters (mm) of mercury (Hg) for about one minute. Particularly effective bubbles retain 75% of the entrapped gas after being exposed for one minute to a pressure of 300 mm Hg, with an entrapped gas content of 90% providing especially effective contrast agents. It is also highly desirable that, after release of the pressure, the bubbles return to their original size. This is referred to generally as xe2x80x9cbubble resiliencexe2x80x9d.
Bubbles which lack desirable stability are poor contrast agents. If, for example, bubbles release the gas entrapped therein in vivo, reflectivity is diminished. Similarly, the size of bubbles which possess poor resilience will be decreased in vivo, also resulting in diminished reflectivity. Accordingly, new and/or better stabilized contrast agents and methods for providing same are needed. The present invention is directed to this, as well as other, important ends.
The present invention provides stable microbubbles created using a lipid shell which encapsulates perfluoropropane gas and in which the stabilizer mPEG-5000 is not covalently bonded to the lipid. Unexpectedly, the gas-filled microbubbles of the present invention in which the stabilizer mPEG-5000 is not covalently bonded to the lipid possess a number of surprising yet highly beneficial characteristics. The microbubble composition of the present invention provides larger microbubble diameter, improved stability and increased opacification when compared to microbubbles prepared using a lipid-mPEG-5000 conjugate. The microbubble composition of the present invention also provides economic benefits because synthesis and purification of the lipid-mPEG-5000 conjugate is not required. The gas-filled microbubbles of the present invention thus have surprisingly and unexpectedly superior characteristics for ultrasound contrast imaging.
Accordingly, one object of the present invention is to provide a composition comprising stable microbubbles in an aqueous solution.
It is another object of the present invention to provide a pharmaceutical composition comprising stable microbubbles in an aqueous solution.
It is another object of the present invention is to provide a process for manufacturing the stable microbubbles.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors"" discovery that a stable microbubbles in an aqueous solution can be conveniently and reproducibly prepared and used for ultrasound.